To elucidate the role of the gonadotropins in the multiple spawner Atlantic halibut (Hippoglossus hippoglossus) full length cDNAs encoding FSHbeta, LHbeta, and the common alpha-subunit were cloned from pituitary glands by RACE-PCR. The three cDNAs consisted of 614, 595, and 666 nucleotides encoding peptides of 131, 146, and 124 amino acids, respectively. Halibut FSHbeta and LHbeta showed unique structural features among the vertebrate glycoprotein hormones. First, in contrast to all known FSHbeta, which contain either one or two conserved N-glycosylation sites, no potential binding site was found in Atlantic halibut FSHbeta. Second, the conserved glycosylation site in the N-terminus of all vertebrate LHbeta has been substituted with a unique C-terminal binding site in Atlantic halibut LHbeta. Furthermore, a specific cysteine residue of importance for the folding and heterodimerization of mammalian FSH is lacking in the FSHbeta from Atlantic halibut as well as many other teleosts. However, teleost FSHbeta is characterized by an additional N-terminal cysteine, which has likely replaced the missing residue, implicating a modified folding pattern of this subunit. In situ hybridization of mature male pituitaries revealed that FSHbeta and LHbeta mRNA were expressed in distinct cell types throughout the proximal pars distalis of the adenohypophysis, while alpha-subunit mRNA was identified in all parts of the proximal pars distalis, and also along the periphery of pars intermedia. Consistently, Northern blot analysis of pituitary RNA from mature males showed that FSHbeta, LHbeta, and alpha-subunit mRNAs were highly expressed. In juvenile male pituitaries very few cells containing FSHbeta, LHbeta, and alpha-subunit mRNA were identified by in situ hybridization. Low mRNA levels encoding LHbeta and the alpha-subunit were also demonstrated by Northern blot analysis of the juvenile pituitaries, while no FSHbeta mRNA was detected using this less sensitive technique. The results suggest that both FSH and LH play a role during both the very early and the final reproductive stages in Atlantic halibut males.